Abstract
BackgroundIt remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies.MethodsWe employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods.ResultsWe demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors.ConclusionsSingle-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.
Highlights
It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies
CD19 CAR-T, arguably the most successful CAR-T cell therapy to date in the clinic, involves genetically engineering autologous T cells ex vivo to express CARs against a B-lineage antigen CD19, which is expressed on tumor cells such as diffuse large B-cell lymphoma (DLBCL) and B-cell precursor acute lymphocytic leukemia (B-ALL) [3,4,5]
Study design - highly multiplexed measurement of cytokine secretions from single CD19 CAR-T cells upon antigen-specific stimulation To capture the full spectrum of complex T cell functions within the heterogeneous CAR-T population, we analyzed CAR-T cytokine production at the single cell level using a 16-plex panel
Summary
It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies. Adoptive immunotherapy is a novel treatment modality for human cancers [1, 2] One such approach, CAR-T cell therapy, involves the use of viral vectors to genetically modify autologous T cells to express a chimeric antigen receptor (CAR) directed against a tumor antigen [2]. An overall response rate of 57% (4 complete responders and 4 partial responders out of 14 patients) was reported in a small trial on relapsed/refractory chronic lymphocytic leukemia (CLL) and a complete response rate of 57% (4 out of 7 patients) was reported in a phase I study with chemotherapy-refractory DLBCL [9] These variable clinical responses across patients may result from many factors such as pre-conditioning regimen of patients, CAR-T cell administration procedure as well as T cell sources and manufacturing process. Highresolution single-cell analysis of functional attributes of CAR-T pre-infusion products may provide new insights into the variability of clinical responses and open up new avenues for the development of more efficacious yet safe CAR-T cell therapy
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